Germine-diacetate in the treatment of myasthenia gravis

ABSTRACT

Germine-3,15-diacetate is prepared by transesterification of germine-3,16-diacetate. This novel compound has been found to be useful in the treatment of myasthenia.

United States Patent [151 3,670,083 Cohen et a]. June 13, 1972 [54]GERMINE-DIACETATE IN THE TREATMENT OF MYASTHENIA GRAVIS Inventors:Edward M. Cohen, Norristown, Pa; Ed-

ward J. J. Grabowski, Westfield, N.J.; Rezso Aczel, Chalfont, Pa.

US. Cl ..424/256, 260/287 Int. Cl. ..A6lk 27/00 Field of Search..424/256; 260/287 References Cited OTHER PUBLICATIONS Chem. Abst.68-94464h, (I968).

Primary ExaminerStanley J. Friedman Attorney-Thomas E. Arther, Harry E.Westlake, Jr. and 1. Louis Wolk 57 ABSTRACT Germine-3, l S-diacetate isprepared by transesterification of germine-B, l G-diacetate. This novelcompound has been found to be useful in the treatment of myasthenia.

2 Claims, No Drawings GERMlNE-DIACETATE IN THE TREATMENT OF MYASTHENIAGRAVIS SUMMARY OF THE INVENTION This invention relates to thepreparation of a new germinediacetate. More particularly, it relates tothe preparation of germine-3,l5-diacetate by the transesterification ofgermine- 3,16-diacetate and to salts and hydrates thereof. Germine-3,15-diacetate has valuable pharmacological properties which provideeffective alleviation of skeletal muscle weakness in patients withmyasthenia gravis.

BACKGROUND OF THE INVENTION The disease known as myasthenia gravis isone which afiects a substantial portion of the population and ischaracterized by fatigue and exhaustion of the muscular system marked byprogressive paralysis of muscles without sensory disturbances oratrophy. It may affect any muscle of the body but especially those ofthe face, lips, tongue, throat and neck. One of the methods used in thepast for the treatment of or the alleviation of the symptoms of thisdisease is the administration of drugs in an attempt to reverse at leasttemporarily some of the wasting affects of the disease. Included amongthe drugs administered was a mixture of veratrum alkaloids. Thisparticular mixture, although it appeared to have some salutary effect,could only be used to a limited extent because of the variety of sideeffects attendant on the administration of the medication.

The compound gennine-3,l5-diacetate is effective in alleviating skeletalmuscle weakness in patients with myasthenia gravis without having theattendant side effects occasioned by the administration of the veratrumalkaloids One might suspect that an ideal manner of preparinggermine-3,l5-diacetate would be by the direct acetylation of the germineitself [5. Morris Kupchan and C. R. Narazanon, .l.A.C.S., 81 zl9l3-2l1959)]. Germine is a complicated polycyclic molecule containing onehetero atom and seven hydroxyl substituents, four of which are readilyacylable. The selective acetylation of the 3- and the -positions is avirtual impossibility without blocking some of the more readily acylablepositions.

We have found that germine-3,l5-diacetate can be conveniently preparedby a transesterification of germine-3,l6- diacetate. A description ofthe preparation of a 3,l6-diester can be found in S. Morris Kupchan andC. R. Narazanon, ibid.

DESCRIPTION AND PREFERRED EMBODIMENTS This invention provides a methodof preparing the novel compound germine-3,l5-diacetate and salts andhydrates thereof. This compound is represented by the followingstructural formula: (3H3 i E In} 000113 on. 0- --on To practice thepresent invention, germine-3,l6-diacetate is converted in good yield togermine-3,l5-diacetate by intrarnolecular transesterification whengermine-3,l6-diacetate is dissolved in aqueous solution above pH 7.

Germine-3,15-diacetate can be prepared by dissolvinggermine-3,16-diacetate in an aqueous solution, the pH of which isgreater than 7. A quantity of organic or inorganic base may be added toeffect the desired pH, however this is not necessary to complete thereaction. Above pH 7, the intramolecular transesterification of thel6-acetoxy group to the l5-acetoxy group occurs. It is preferable to usereaction conditions where the pH of the medium is 7-10 since at pHgreater than this further reaction may occur. Such bases that may bepresent are 2,6-lutidine, s-collidine, pyridine, ammonium hydroxide, aninorganic bufi'er having desired pH such as a phosphate buffer, etc.

The reaction must be carried out in the presence of water which enablesthe transesterification of the lo-ester to 15- ester group to proceed.At least a catalytic amount of water should be present in order to formthe desired compound but higher quantities can be used. It is preferableto use about a 50 percent aqueous medium. It is most desirable to runthe reaction in a ratio of water and solvent in which the genuine-3,16-diacetate is appreciably soluble. The amount of water present couldlikewise be adjusted for the convenience of the reaction and to suitother reaction conditions such as temperature, time, pH, etc. Ifpreferred, this reaction may further be carried out in an entirelyaqueous medium, the pH of which would depend on the amount ofgermine-3,l6-diacetate present.

The reaction may also be carried out in the presence of a solvent. It isbest to use a solvent which is miscible with water since water is alsonecessary for the completion of the reaction. It is also preferable tochoose a solvent in which the germine-3,l6-diacetate is soluble. Thesolvent may likewise be the basic medium itself which is diluted withwater (such as 50 percent aqueous pyridine or a phosphate bulTer havinga pH of about 8). Representative solvents which can be used alone or incombination include acetone, ethanol, methanol, pyridine, acetonitrile,dimethylsulfoxide, nitromethane, dimethylformamide, dimethoxyethane,etc.

The temperature of the reaction employed and the time necessary to carryout the reaction will depend on the various other reaction conditionspresent. It is preferred to carry out the reaction at a temperaturebetween about 040 C., and 20-30 C. being most preferable. The reactionmay be carried out from less than 1 hour to several weeks and stillobtain germine-3, l 5-diacetate.

The separation and purification of germine-3,l5-diacetate can be carriedout by methods known in the art. It is preferred and convenient toaccomplish this separation and purification of genuine-3,15-diacetate bychromatographic techniques, one such system being a silica gel columnusing 5-95 percent ethanol in chloroform as the eluant. It is furtherconvenient to follow the extent of the conversion of the reaction byascending thin-layer chromatography using a mobile ethylacetatemethanol-concentrated ammonia 15:5) eluant with silica gel plate.

Gem1ine-3,l5-diacetate may be readily converted to its non-toxic acidaddition salts by customary methods in the art; such salts would includethose prepared from hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, methanesulfonic acid, acetic acid,propionic acid, oxalic acid, glycolic acid, lactic acid, salicylic acid,etc. It is possible to form a hydrate and separate this from thereaction mixture; thus, for example, germine-3,l5-diacetate oxalatemonohydrate may be prepared by titrating germine-3,l5- diacetate with anaqueous solution of oxalic acid and isolating the desired hydrate bymethods known in the art. These hydrates are included within the scopeof this invention.

Germine-3,15-diacetate, its non-toxic salts, and the hydrates thereof,are efi'ective in increasing the tension of skeletal muscle in responseto motor nerve stimulation. The

neuromuscular actions are similar to those obtained with other veratrumalkaloids, however it differs in that it lacks hypotensive and emeticproperties common to the veratrum series.

Various tests in animals can be carried out to show the ability ofgermine-3,l5-diacetate to exhibit reactions that can be associated withskeletal muscle effect in humans. One such test determines the tensionresponses of the gastrocnemius and soleus muscles to stimulation of thesciatic nerve in anesthetized cats. Data is expressed as the ratio ofthe twitch response to a single stimulus delivered at a frequency of 24per minute to the maximum response to a IO-second tetanic stimulusdelivered at a frequency of 50 cps. The responses are recorded followingsingle dose administration. This test is outlined by Werner Flacke inthe .1. Pharm. and Exp. Ther., 1412230-236 1963). In view of resultsfrom tests such as this, germine-3,l5-diacetate can be expected to beeffective in improving the skeletal-muscle power of patients withmyasthenia.

Germine-3,l5-diacetate and its pharmaceutically acceptable salts can benormally administered orally, parenterally or rectally. The termparenteral as used herein includes subcutaneous injection, intravenous,intramuscular or intrasternal injection or infusion techniques.

Orally, they may be administered in tablets, capsules, suspensions orsyrups, the optimum dosage depending, of course, on the particularcompound being used and the severity of the condition being treated. inany specific case, the appropriate dosage selected will further dependon factors of the human patient which may influence response to thedrug, for example, general health, age, weight, etc. Although theoptimum quantity of germine-3,15-diacetate to be used in such mannerwould depend on the severity of the condition treated, oral dose levelsin the range of 025-300 mg./kg. (preferably in the range of 260 mg./kg.per day) show excellent effectiveness. Comparative dosages may be usedin parenteral or rectal administration.

Compositions may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions and such compositionsmay contain one or more agents; for example, sweetening agents,flavoring agents, coloring agents, preserving agents, etc. Further,germine-3, l 5- diacetate or its salts may be administered alone or inadmixture with other active ingredients such as anticholinesteraseagents and/or non-toxic pharmaceutically acceptable excipients. Suchexcipients may be, for example, inert diluents such as calcium carbonateand lactose; granulating and disintegrating agents, for example, cornstarch and alginic acid; lubricating agents, for example, magnesiumstearate and talc; binding agents, for example, starch and gelatin;suspending agents, for example, methylcellulose and vegetable oil;dispersing agents, for example, lecithin; thickening agents, forexample, beeswax and hard paraffin; emulsifying agents, for example,naturally-occurring gums; and non-irritating excipients, for example,cocoa butter and polyethylene glycols. Although the unit dosage ofgermine-3, l S-diacetate to be used would depend on factors of thepatient, unit dosages contain ing between about 1-100 mg. are consideredto be useful.

The following examples show the preparation of germine- 3,15-diacetateand are to be construed as illustrations of this invention and not aslimitations thereof.

A solution of 10 g. of germine-3,l6-diacetate in 200 ml. of 50 percentaqueous pyridine is allowed to stand at room temperature for threeweeks. After addition of 50 ml. of concentrated ammonium hydroxide it isextracted with three ml. portions of chloroform, dried over magnesiumsulfate, tiltered, concentrated at reduced pressure and dried at 100 invacuo to afiord 8.1 g. of amorphous yellowish solid. Drycolumnchromatography (1 g. of mixture to 200 g. of silica gel) using 10percent ethanol in chloroform as eluant is used to obtain the free baseof germine-3,l5-diacetate as an amorphous solid.

The above reaction, when carried out for 1 hour in a methanol-watersolution 1:1 by volume) and adjusting the pH to 9.5 with 0.1 M aqueousammonium results in germine-3,l5- diacetate.

Germine-fl, l S-diacetate is titrated in methanol with an oxalic acidsolution to pH 5. After removal of the methanol, the residue isrecrystallized from acetonitrile-ether to afford germine-3,15-diacetateoxalate monohydrate (m.p 2 l4216, dec.). Anal. calcd. for C H NO, (mol.wt. 701.77)

C, 56.48; H, 7.33; N, 2.00.

Found:

Representative composition preparations are as follows:

A solution of 200 mg. (0.34 mmoles) of germine-3,l5- diacetate in 100ml. of water is prepared and 23 mg. of anhydrous citric acid (0.12mmoles) is added to this with stirring. This is then divided into 10 ml.aliquots, each containing 20 mg. of germine-3, l S-diacetate for use ininjectables.

Capsules for oral use, each containing 50 mg. of germine- 3,15-diacetateare prepared by blending 50 g. of germine- 3,15-diacetate with 237 g. oflactose U.S.P. and 4.10 g. of magnesium stearate. This is then used tofill 1,000 capsules each containing 50 mg. of germine-3, l S-diacetate.

What is claimed is:

2. A pharmaceutical composition for the treatment of myasthenia graviswhich comprises as active ingredient 1-100 mg. ofgermine-3,15-diacetate, a non-toxic pharmaceutically acceptable salt,thereof or the hydrate thereof.